Methods and compositions for generating mitochondria replaced lymphoid cells

Abstract

The present disclosure provides methods and compositions for generating mitochondria replaced lymphoid cells, such as T cells, that involves incubating lymphoid cells that have not undergone a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of mammalian target of rapamycin (mTOR) inhibitor. In addition, the present disclosure also provides methods of treating an immunological deficiency associated with heteroplasmic immune cells, as well as methods for ameliorating a symptom of mitochondrial complex III deficiency, that involve administering the mitochondria replaced lymphoid cells.

What is claimed is: 
     
         1 . A method for generating mitochondria replaced lymphoid cells in which at least 20% of endogenous mitochondrial DNA (mtDNA) has been replaced with exogenous mtDNA, wherein the method comprises
 incubating lymphoid cells that have not undergone a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer the exogenous mitochondria to the lymphoid cells, thereby generating mitochondria replaced lymphoid cells in which at least 20% of the endogenous mtDNA has been replaced with exogenous mtDNA.   
     
     
         2 . The method of  claim 1 , wherein the mTOR inhibitor comprises rapamycin or a derivative thereof. 
     
     
         3 . The method of  claim 1 or claim 2 , wherein the mTOR inhibitor is rapamycin. 
     
     
         4 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 100 nM to about 1000 nM. 
     
     
         5 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 200 nM to about 500 nM. 
     
     
         6 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 100 nM. 
     
     
         7 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 200 nM. 
     
     
         8 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 500 nM. 
     
     
         9 . The method of any one of  claims 1 to 3 , wherein the effective amount of the mTOR inhibitor is a concentration of about 1000 nM. 
     
     
         10 . The method of any one of  claims 1 to 9 , wherein the mitochondria replaced lymphoid cells comprises at least 20% of exogenous mtDNA and no more than 80% endogenous mtDNA, as measured by TaqMan Single Nucleotide Polymorphism (SNP) Assay. 
     
     
         11 . A method for generating mitochondria replaced lymphoid cells, wherein the method comprises incubating lymphoid cells that have not undergone a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and about 100 nM to about 1000 nM of rapamycin for a sufficient period of time to non-invasively transfer the exogenous mitochondria to the lymphoid cells, thereby generating a mitochondria replaced lymphoid cells. 
     
     
         12 . The method of any one of  claims 1 to 11 , wherein the isolated exogenous mitochondria is about 20 μg to 80 μg protein per 1×10 6  cells. 
     
     
         13 . The method of any one of  claims 1 to 12 , further comprising centrifuging the lymphoid cells prior to incubating. 
     
     
         14 . The method of  claim 13 , wherein centrifuging is performed at 1,500 relative centrifugal force (RCF) for approximately 5 minutes at room temperature. 
     
     
         15 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is at least approximately 24 hours. 
     
     
         16 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is at least 36 hours. 
     
     
         17 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is at least 48 hours. 
     
     
         18 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is approximately 2 days or more. 
     
     
         19 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is approximately 7 days or more. 
     
     
         20 . The method of any one of  claims 1 to 14 , wherein the sufficient period of time is approximately 2 days to approximately 7 days. 
     
     
         21 . A method for generating mitochondria replaced lymphoid cells, wherein the method comprises:
 (a) centrifuging lymphoid cells and isolated exogenous mitochondria under conditions sufficient to generate a cell pellet, wherein the lymphoid cells have not undergone a procedure to reduce or deplete endogenous mitochondria; and   (b) incubating the lymphoid cells with 100 nM to 1000 nM of rapamycin for approximately 24 hours or more, thereby generating mitochondria replaced lymphoid cells.   
     
     
         22 . The method of  claim 21 , wherein incubating is for approximately 7 days or more. 
     
     
         23 . The method of  claim 21 , wherein incubating is for approximately 2 days to approximately 7 days. 
     
     
         24 . The method of any one of  claims 1 to 23 , wherein the lymphoid cells are T cells, B cells, monocytes, macrophages, natural killer (NK) cells, or granulocytes. 
     
     
         25 . The method of  claim 24 , wherein the lymphoid cells are T cells. 
     
     
         26 . The method of  claim 25 , wherein the T cells comprise exhausted T cells, senescent T cells, or a combination thereof. 
     
     
         27 . The method of any one of  claims 1 to 26 , wherein the lymphoid cells are human lymphoid cells. 
     
     
         28 . A composition comprising an effective amount of the mitochondria replaced lymphoid cells generated by the method of any one of  claims 1 to 27 , and a pharmaceutically acceptable carrier. 
     
     
         29 . A method for ameliorating a symptom of mitochondrial complex III deficiency to a subject in need thereof, comprising administering to the subject the composition of  claim 28 . 
     
     
         30 . A method for treating an immunological deficiency associated with heteroplasmic immune cells to a subject in need thereof, comprising administering to the subject the composition of  claim 28 . 
     
     
         31 . The method of  claim 30 , wherein the subject has received a reverse transcriptase inhibitor. 
     
     
         32 . The method of  claim 31 , wherein the subject has human immunodeficiency virus (HIV). 
     
     
         33 . The method of  claim 31 , wherein the subject has hepatitis B virus (HBV). 
     
     
         34 . The method of any one of  claims 29 to 33 , wherein the subject is human.